Detection of Isocitrate Lyase in Achlya Flagellata

Proceedings of the Iowa Academy of Science

Volume 81 Number Article 7

1974

Detection of Isocitrate Lyase in Achlya flagellata

Joanne Tontz Ellzey University of Texas at El Paso

Christina D. Chavez University of Texas at El Paso

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Recommended Citation Ellzey, Joanne Tontz and Chavez, Christina D. (1974) "Detection of Isocitrate Lyase in Achlya flagellata," Proceedings of the Iowa Academy of Science, 81(1), 7-9. Available at: https://scholarworks.uni.edu/pias/vol81/iss1/7

This Research is brought to you for free and open access by the Iowa Academy of Science at UNI ScholarWorks. It has been accepted for inclusion in Proceedings of the Iowa Academy of Science by an authorized editor of UNI ScholarWorks. For more information, please contact [email protected]. Ellzey and Chavez: Detection of Isocitrate Lyase in Achlya flagellata

Detection of Isocitrate Lyase in Achlya flagellata

JOANNE TONTZ ELLZEY and CHRISTINA D. CHAVEZ1

ELLZEY, JOANNE ToNTZ, and CHRISTINA D. CHAVEZ (Dept. of Achlya flagellata oogonial cultures. This enzyme is an indicator of Biological Sciences, University of Texas at El Paso, El Paso, Texas the glyoxylate pathway (metabolism of lipids to carbohydrates). 79968). Detection of Isocitrate Lyase in Achlya flagellata. Proc. Further work is in progress to determine if there is a correlation IowaAcad. Sci. 81(1): 10-11, 1974. between isocitrate lyase and the microbodies of oogonial initials. The colorimetric procedure of McFadden and Howes ( 1960) and INDEX DESCRIPTORS: Isocitrate Lyase, Achlya flagellata, Achlya the enzyme assay for isocitrate lyase ( Shiio, Shiio, and McFadden, lsocitrate Lyase. 1965) gave positive results for the presence of isocitrate lyase in

The glyoxylate cycle couples fatty acid degradation with day-old cultures containing hyphae, oogonia, antheridia, and carbohydrate synthesis. C2 compounds enter the citric acid oospores were harvested by centrifugation at 15,000 rpm for cycle as acetyl-CoA, which condenses with oxaloacetic acid, 15 min. A mortar and pestle were used to disrupt the fungal producing citric acid. The citric acid is converted to isocitric walls in 0.05 M Tris buffer containing 0.05 mM MgCl~. After acid. The key enzymes of the glyoxylate cycle are ( 1) iso citrate lyase, which catalyzes the conversion of isocitrate ~ succinate and glyoxylate and ( 2) malate synthase, which 7 catalyzes the reaction, acetyl coenzyme A and glyoxylate ~ malate (Dixon and Kornberg, 1959). Reports of isocitrate lyase in fungi have been primarily concerned with the higher fungi (Kornberg and Collins, 1958; Collins and Kornberg, 1960; Gottlieb and Ramachan dran, 1960; Heberling, Berky, and Stone, 1960; Frear and E Johnson, 1961; Turian, 1961; Duntze et al., 1969; Kohr, Van c: derhaeghe, and Combepine, 1969; Szabo and Avers, 1969; 0 C\I Cotter et al., 1970; Perlman and Mahler, 1970; Selem and I() 1.5 Sawsan, 1970; O'Sullivan and Casselton, 1973). McCurdy I- and Cantino ( 1960) discovered a metabolic route from gly <( oxylate to glycine in the chytridiomycete Blastocladiella em >- ersonii. The following paper is the first report of the gly I- (/) oxylate pathway in the oomycetes. z In unpublished ultrastmctural data, Ellzey observed the LLI c association of microbodies with storage bodies. The contents 1.0 of the storage bodies appeared to be converted from lipid to _J carbohydrate in Achlya flagellata Coker oogonial initials. <( Therefore, an assay was performed to determine if isocitrate (.) I- lyase was present in oogonial cultures of A flagellata. a.. 0

MATERIALS AND METHODS 0.5 +EXP Pure cultures of Achlya flagellata (ATCC #14566) were oCONTROL grown on autoclaved hemp seeds in sterile water, as well as on Barksdale Medium #5, containing 400 mg edamin, 2400 mg glucose, 80 mg calcium glycerophosphate, 1 ml 0.5 M MgS04 • 7H~O, 0.5 ml 2.0 M KCl, 0.5 ml metal mix #4, and 100 mg Tris in 1000 ml distilled water (pH 6.8). After 0 autoclaving, 400 mg of potassium bicarbonate in 10 ml of 2.0 4.0 6.0 8.0 distilled water was added to Barksdale Medium #5. Five- X 10-2 mg GLYOXYLATE

1 Department of Biological Sciences, University of Texas at El Figure 1. Optical density at 520 nm as a function of glyoxylate Paso, El Paso, Texas 79968. concentration in 5 ml of solution.

Published by UNI ScholarWorks, 1974 1 Proceedings of the Iowa Academy of Science, Vol. 81 [1974], No. 1, Art. 7

ISOCITRATE LYASE IN ACHLYA FLAGELLATA 11

centrifugation at 15,000 rpm for 15 min, the supernatant REFERENCES CITED from 1 gm wet wt. of fungus was assayed for isocitrate ly ase, using the colorimetric procedure of McFadden and Howes ( 1960) and Shiio, Shiio, and McFadden ( 1965). The CoLLINS, J. F., and H. L. KORNBERG. 1960. The metabolism of C2 supernatant was preincubated with 55 p.moles of glutathione compounds in micro-organisms. Biochem. ]. 77: 430-438. for 10 min at room temperature in a total volume of 5 ml COTTER, D. A., A. J. LACLAvE, W. S. WEGENER, and D. J. NEI containing 455 p.rnoles of Tris and 13.6 p.moles of MgCl~. DERPRUEM. 1970. C02 control of fruiting in Schizophyllum To initiate the enzymatic reaction, 0.45 ml of 0.04 M sod commune: Noninvolvement of sustained isocitrate lyase dere ium DL-isocitrate was added and allowed to react for IO pression. Can. ]. Microbial. 16: 605-608. min at 30° and 20 min at room temperature. The reaction D1xoN, G. H., and H. L. KORNBERG. 1959. Assay for key enzymes was stopped with the addition of 2.3 ml of 10% (w/v) tri of the glyoxylate cycle. Biochem. ]. 72: 3P. chloracetic acid. The fungal reaction mixture was assayed DuNTZE, W., D. NEUMANN, J.M. GANCEDO, W. ATZOPODIEN, and H. HOLZER. 1969. Studies on the regulation and localization of for the formation of glyoxylic acid phenylhydrazone from the glyoxylate cycle enzymes in Saccharomyces cerevisiae. Eu glyoxylate, an endproduct of ropean ]. Biochim. 10: 83-89. isocitrate lyase FREAR, D. S., and M. A. JoHNSON. 1969. Enzymes of the glyoxylate isocitrate ~------~ succinate and glyoxylate cycle in germinating uredospores of M elampsora lini ( pers.) lev. and phenylhydrazine hydrochloride. Biochem. Biophys. Acta 41: 419-421. The control consisted of a 5 ml solution containing 0.025- GOTTLIEB, D., and S. RAMACHANDRAN. 1960. The nature of pro 0.2 p.moles glyoxylate. One ml of 1 M oxalic acid buffer at duction of the glyoxylate pathway enzymes in germinating pH 1 and 1.0 ml of 1% phenylhydrazine HCl were added to spores of Penicillium oxalicum (F-56). Mycol.ogia 52: 599-607. the control. This solution was boiled and then cooled for 5 HEBERLING, R. L., J. J. BERKY, and R. W. STONE. 1960. Dissimila tion of citric acid by extracts of Penicillium chrysogenum. Arch. min. The control was then chilled in an ice bath to room Biochem. Biophys. 86: 102-109. temperature. Concentrated HCl ( 4 ml) and 5% potassium KoBR, M. J., F. VANDERHAEGHE, and G. CoMBEPINE. 1969. Particu ferricyanide ( 1.0 ml) were added. After thorough mixing, late enzymes of the glyoxylate cycle in Neurospora crassa. Bio the percent transmission vs. p.moles of glyoxylate was deter chem. Biophys. Res. Comm. 37: 640-645. mined using a Bausch and Lomb Spectronic 20 colorimeter KORNBERG, H. L., and J. F. COLLINS. 1958. The glyoxylate cycle set at 520 nm. This wavelength was chosen after plotting a in Aspergillus niger. Biochem. ]. 68: 3P-4P. spectrum for the red-violet colored product of oxidized gly McCuRDY, H. D., JR., and E. C. CANTINO. 1960. lsocitratase, oxylic acid phenylhydrazone and selecting the peak absorb glycine-alanine transaminase, and development in Blastocladiella ancy. The same procedure was followed for 5 ml of the emersonii. Plant Physiol. 35: 463-476. fungal supernatant. McFADDEN, B. A., and W. V. HowEs. 1960. The determination of glyoxylic acid in biological systems. Anal. Biochem. 1: 240-248. O'SULLIVAN, J., and P. J. CASSELTON. 1973. The subcellular local ization of glyoxylate cycle enzymes in Coprinus lagopus ( sensu Buller). J. Gen. Microbial. 75: 333-337. RESULTS AND DISCUSSION PERLMAN, P. S., and H. R. MAHLER. 1970. Intracellular localization of enzymes in yeast. Arch. Biochem. Biophys. 136: 245-259. SELEM, K. G., and A. E. SAWSAN. 1970. The presence of isocitrate Figure 1 compares the optical density of the control con lyase in Aspergillus niger. ]. Microbial. UAR. 3: 233. taining 0.025-0.2 p.moles glyoxylate/5 ml solution with the Smm, I., T. Smm, and B. A. McFADDEN. 1965. lsocitrate lyase optical density of 5 ml fungal supernatant. A red-violet pro from Pseudomonas indigofera. I. Preparation, amino acid com duct was obtained from five-day-old cultures of A. flagellata position and molecular weight. Biochim. Biophys. Acta 96: 114- grown on either hemp seeds or Barksdale Medium #5. Ex 122. perimental solutions had 9.8 and 6. 7% transmission. These SzABO, A. S., and C. J. AvERs. 1969. Some aspects of regulation of peroxisomes and mitochondria in yeast. Ann. N. Y. Acad. Sci. readings were within the control range of 2.2-64.5% trans 168: 302-312. mission. TURIAN, G. M. 1961. L'acetate et son double effet d'induction iso The positive assay for isocitrate lyase in A. flagellata indi citratasique et de differenciation conidienne chez les Neurospora. cates the presence of the glyoxylate pathway. Further work C. R. Acad. Sci. (Paris), ser. D, 252: 1374-1376. is in progress to determine if microbodies of oogonial init ials contain isocitrate lyase. Ultimately, we seek to determine if storage bodies of oogonial initials are the sites of conversion of lipids to carbohydrates.

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